Insulin resistance and type 2 diabetes (T2D) represent a global health crisis of potentially disastrous economic impact. In the United States alone, over one hundred million individuals suffer from obesity, the leading risk factor for pre-diabetic and diabetic syndromes. Recently, there has been widespread interest in treating obesity, T2D, and associated metabolic disorders by enhancing the activity of brown fat, a tissue capable of burning excess lipid stores via uncoupled -oxidation. At present, however, the regulation of brown fat metabolism is incompletely understood, and it is unclear how this tissue exerts its therapeutically promising effects on systemic energy homeostasis. This proposal aims to advance the understanding of fundamental brown fat physiology by interrogating the role of the mitochondrial calcium uniporter, a channel whose molecular identity was recently elucidated by the applicant's laboratory. The uniporter couples ATP production to cellular energy demand, and may play a key role in fuel switching through its ability to regulate TCA cycle flux and anaplerosis. The uniporter is linked to adrenergic signaling in multiple tissues and supports peak bioenergetics in skeletal muscle, a close developmental and metabolic relative of brown fat; in addition, uniporter current density in brown fat mitochondria i among the highest measured in any tissue. The uniporter is therefore a promising candidate to regulate brown fat metabolism in response to hormonal stimuli. The applicant will pursue the core hypothesis that the uniporter plays a central role in brown fat physiology in two complementary but not codependent specific aims. In Aim 1, the uniporter's pore- forming subunit (MCU) will be ablated in immortalized brown adipocytes, and the resulting cell lines will be analyzed for defects in bioenergetics, metabolism, stress signaling, and calcium dynamics. Exciting preliminary data indicate that brown adipocytes lacking MCU exhibit blunted catecholamine-stimulated respiration; these results will be rigorously validated and dissected mechanistically. In Aim 2, MCU will be deleted in brown fat in mice, and the resulting animals will be assayed for altered energy expenditure and brown fat activity, cold intolerance, increased susceptibility to diet-induced obesity, and insulin resistance. The applicant's laboratory has already generated mice harboring a conditional MCU allele that can be used to ablate uniporter activity in a tissue-specific manner. By establishing a role for the uniporter in brown fat, this wrk will yield foundational insights into the physiology of this tissue, laying the groundwork for its clinical exploitation to ameliorate T2D and obesity. The work will also complement the proposed training plan, enabling the applicant to develop into a mature, rigorous scientist with the abilityto communicate across a variety of topical disciplines.